Thermostable collagen-digesting enzyme, novel microorganism producing the enzyme and process for producing the enzyme

ABSTRACT

Bacillus sp. NTAP-1 having been deposited under accession number FERM BP-6926; and a collagen-decomposing enzyme produced by bacterium. The above enzyme (1) has a capability of hydrolyzing, at the highest efficiency, collagen and gelatin from among casein, gelatin, albumin and collagen; (2) shows the optimum pH of 3.5 to 4.5; (3) shows the optimum temperature of 65 to 70° C.; (4) after heating at 60° C. at pH 6.0 for 4 hours, sustains an activity amounting to 60% or more of the level before the heat treatment; (5) remains stable at pH 3 to 6; and a molecular weight of approximately 46,000 when measured by SDS-PAGE.

FIELD OF THE INVENTION

The present invention relates to a thermostable collagen-decomposingenzyme produced by a new microorganism, and said new microorganism and amethod for production of said enzyme by said microorganism. Concretely,the present invention relates to a novel thermostablecollagen-decomposing enzyme having the highest reactivity (substratespecificity) to collagen, produced by a novel microorganism of the genusBacillus, said novel microorganism and a process for producing of saidenzyme by said microorganism.

DESCRIPTION OF THE PRIOR ART

Collagen-decomposing and gelatin-decomposing enzymes have been widelyused in industry. For example, collagen peptides, which are hydrolyticproducts from collagen by these enzymes, are useful as material forcosmetics, because of their interesting physiological activities such asmoisture-keeping effects or immunity-activation activity. Therefore,collagen peptides are widely used for medical and cosmetic purposes.Further, gelatin, which is a denaturated form of collagen, is used asthe coating material for photograph films, and gelatin-decomposingenzymes are used for the recycling of the photograph and X-ray films.Many kinds of proteases are known to decompose difficult. For hydrolysisof collagen, specific metal proteases, named “collagenase”, should beused.

Recently, many attempts have been made for the effective use of theorganic wastes. For instance, the composing of garbage wastes is oneconcrete example of the biorecycling of organic wastes. More than 30% ofanimal protein are composed of collagen, therefore, garbage wastesproduced from daily kitchen activities in houses and restaurants and thewastes from meat processing factories should contain large quantities ofcollagen.

Because of specific, highly ordered structure, collagen is generallyinsoluble in water and difficult to be decomposed, and thereforedegradation of collagen proceeds very slowly during composing. Most partof unusable portions produced from livestock industries are composed ofcollagen, and, therefore, are treated by incineration, causing problemssuch as anathermal of the earth or the generation of carbon dioxide ordioxin which cause the air pollution.

These problems must be solved from the view point to make an effectiveuse of materials.

It is well known that the temperature of the organic wastes raises to50-65° C. or higher during the composing process. Therefore, ifthermostable enzymes or thermophilic microorganisms which are activeeven under such high-temperature composing conditions are used, thecomposing of organic waste should proceed more effectively.

Nowadays, industrial collagenases are those from microorganisms(bacteria), and as a concrete example, an actinomycetous collagenase ofthe genus Streptomyces can be mentioned. Other microbial collagenasesare also known; for instance, collagenases from Clostridium hystolyticum(Biochemistry 1984, 23, 3077-3085) and Cytophaga sp. (Biosci, Biotech,Biochem., 1993, 57, 1894-1898) are the concrete examples.

Concerning the example of enzyme which is industrially used, it isnecessary for the enzyme to be thermostable from the view point oftreating speed and the subject to be treated.

However, all known collagenases are of mesophilic origin and lacks ofthermostability, and these circumstances hampers their efficientindustrial applications. Until now, a collagen-decomposing enzyme withsufficient thermostability (having high optimum temperature) for theindustrial applications has not yet been developed.

Usually, it is difficult to use collagen in an industrial scale becausethere is no thermostable enzyme to act effectively in an industrialscale. Therefore, it is obvious that above mentioned problem can besolved perfectly, if an enzyme having high activity to collagen isdeveloped.

As mentioned above, the object of this invention is to find out athermostable collagen-decomposing enzyme.

The inventors of this invention have carried out an intensive study tofind a microorganism producing a thermostable collagen-decomposingenzyme in nature, and have found a promising thermophilic bacteriumbelonging to the genus Bacillus genus that produces said enzyme in thesoil of Sendai, Japan and accomplished the present invention.

The microorganism, which is used to produce a thermostablecollagen-decomposing enzyme of this invention, belongs to the genusBacillus, and is termed strain NTAP-1. This strain has been depositedaccording to the requirement of deposit based on Budapest treaty in theBiotic Technology Industries Institute of the Agency of IndustrialScience of Technology belonging to the Ministry of International Tradeand Industry Japan and accepted by the accept number of FERM BP-6926 onNov. 1, 1999 Biotic Technology Industries Institute, Agency ofIndustrial Science and Technology 1-3, Higashi 1-chome Tsukuba-shi,Ibaraki-ken 305-0046 JAPAN. (This strain is originally deposited on Aug.27, 1999 under accession number FERM P-17535.) (in the specification,this strain is shortened only as <NTAP-1 strain>)

The inventors of this invention have found out that the industriallyuseful enzyme can be obtained by the use of this strain, and theobtained enzyme can be used as the catalyst for bioconversion.

DETAILED DESCRIPTION OF THE INVENTION

The first important point of this invention is a thermostablecollagen-decomposing enzyme obtained by the microorganism having athermostable collagen-decomposing activity and belonging to the genusBacillus, which is characterized by the following features; (1) thebacterium is Gram-negative or Gram-indefinite, (2) the bacterium has aspore forming ability, (3) the bacterium is motile, (4) the bacteriumgrows at 70° C., does not grow at 30° C. or 80° C. and grows at pH 5,does not grow at pH 7, (6) the bacterium is rod-shaped, (7) thebacterium is negative to catalase, (8) the bacterium is negative tooxidase, (9) the bacterium is negative to O/F test, (10) the bacteriumhas acetoin producing activity and (11) the bacterium has gelatindecomposition activity. Accordingly, an excellent action and effectwhich can be used for the decomposition of collagen at 70° C. or lowertemperatures can be expected.

Desirably, said thermostable collagen-decomposing enzyme of thisinvention is characterized by the following features: (1) the enzyme canfar more effectively hydrolyze collagen and gelatin than casein andalbumin, (2) optimum reaction pH is between pH 3.5 and 4.5, (3) optimumreaction temperature is between 65° C. and 70° C., (4) the enzymeretains more than 60% of its original activity after heat treatment at60° C. and pH 6.0 for 4 hours, (5) the enzyme is stable between pH 3 to6 and (6) molecular weight of the enzyme estimated by SDS-polyacrylamidegel electrophoresis is approximately 46,000. And more desirably, saidthermostable collagen-decomposing enzyme of this invention is producedby the microorganism belonging to the genus Bacillus or Bacillus sp.strain NTAP-1.

The second important point of this invention is a producing method ofthe thermostable collagen-decomposing enzyme comprising, using amicroorganism which has following features, that is, by the followingfeatures: (1) the bacterium is Gram-negative or Gram-indefinite, (2) thebacterium has a spore forming ability, (3) the bacterium is motile, (4)the bacterium grows at 70° C., does not grow at 30° C. or 80° C. andgrows at pH 5, does not grow at pH 7, (6) the bacterium is rod-shaped,(7) the bacterium is negative to catalase, (8) the bacterium is negativeto oxidase, (9) the bacterium is negative to O/F test, (10) thebacterium has acetoin producing activity and (11) the bacterium hasgelatin decomposition activity, purifying and accumulating thethermostable collagen-decomposing enzyme which has following features:(1) the enzyme can far more effectively hydrolyze collagen and gelatinthan casein and albumin, (2) optimum reaction pH is between pH 3.5 and4.5, (3) optimum reaction temperature is between 65° C. and 70° C., (4)the enzyme retains more than 60% of its original activity after heattreatment at 60° C. and pH 6.0 for 4 hours, (5) the enzyme is stablebetween pH 3 to 6 and (6) molecular weight of the enzyme estimated bySDS-polyacrylamide gel electrophoresis is approximately 46,000, in aculture medium and by collecting it.

Desirably, the producing method of said thermostablecollagen-decomposing enzyme, wherein the microorganism belonging to thegenus Bacillus is the Bacillus genus bacteria NTAP-1 strain.

The third important point of this invention is a new developedmicroorganism belonging to a Bacillus genus, which produces saidthermostable collagen-decomposing enzyme, desirably, said microorganismis the strain titled as Bacillus sp. NTAP-1 and have deposited accordingto the requirement of deposit based on Budapest treaty in the BioticTechnology Industries Institute of the Agency of Industrial Science ofTechnology belonging to the Ministry of Intentional Trade and IndustryJapan and accepted by the accession number FERM BP-6926 on Nov. 1, 1999Biotic Technology Industries Institute, Agency of Industrial Science andTechnology 1-3, Higashi 1-chome Tsukuba-shi, Ibaraki-ken 305-0046 JAPAN.(This strain is originally deposited on Aug. 27, 1999 under accessionnumber FERM P-17535.).

The inventors of this invention have found that among the microorganismbelonging to Bacillus genus there is a novel microorganism whichproduces thermostable collagen-decomposing enzyme, and have accomplishedthe present invention.

BRIEF ILLUSTRATION OF THE DRAWINGS

FIG. 1 is a graph showing the heat stability of the thermostablecollagen-decomposing enzyme,

FIG. 2 is a graph showing the pH-stability of said thermostablecollagen-decomposing enzyme,

FIG. 3 is a graph showing the temperature-dependence of the reaction ofsaid thermostable collagen-decomposing enzyme and

FIG. 4 is a graph showing the pH-dependence of the reaction of saidthermostable collagen-decomposing enzyme.

THE BEST EMBODIMENT OF THE INVENTION

The present invention will be illustrated more in detail.

A. The Microbiological Features of Bacteria used to Produce aThermostable Collagen-decomposing Enzyme are Mentioned above. Further,this Microorganism can be Preserved by Freezing Method (−80° C. around).

B. Growing Condition

name of cultivate medium: GGY medium

components of medium: medium containing 1.5% of glucose, 1.5% of gelatinand 0.01% of yeast extract.

pH of medium: 4.8

sterilizing condition of medium: 20 minutes at 120° C.

temperature of medium: 60° C.

aerobic condition

C. Component of Protecting Agent: 30% Glycerol Aqueous Solution (notNecessary to Adjust pH of Protecting Agent)

not necessary to adjust pH of protecting agent

sterilizing condition of protecting agent: 20 minutes at 120° C.

The characteristics of the thermostable collagen-decomposing enzyme ofthis invention will be illustrated more minutely with reference to thedrawings.

FIG. 1 is the graph showing the relative remaining activity of thethermostable collagen-decomposing enzyme after heat treatment at varioustemperature for 1 hour, and in this graph, the activity after heattreatment at 30° C. is taken to be 100%.

FIG. 2 is the graph showing the relative remaining activity of thethermostable collagen-decomposing enzyme after treatment at various pHsfor 1 hour, and in this graph, the activity after treatment at pH 4.1 istaken to be 100%.

FIG. 3 is the graph showing the relative activity of the thermostablecollagen-decomposing enzyme at various temperature, and in this graph,the enzyme activity at 60° C. that indicates maximum activity is takento be 100%.

FIG. 4 is the graph showing the relative activity of the thermostablecollagen-decomposing enzyme at various pHs, and in this graph, theenzyme activity at pH 3.8 that indicates maximum activity value is takento be 100%.

EXAMPLES Example 1

Various kinds of specimen such as soils, composts, river and lake watersare diluted to 100-10,000 times with 0.85% NaCl, and 0.1 ml of saiddiluted solution was spread on GGY agar-agar medium, then are left for 2or 3 days at 70° C. The colony grown on medium was isolated andinoculated in 5 ml of GGY liquid medium and cultivated with shaking for2 or 3 days at 70° C. The collagen-decomposing enzyme activity ofseveral hundred kinds of isolates are evaluated using the supernatant ofculture according to the method described in Example 2.

The strain that indicates the highest collagen-decomposing activity wasselected and named it NTAP-1 strain.

The taxonomical characteristics of NTAP-1 strain can be illustrated asfollows.

(1) cell morphology: rod-shaped (0.8×2-3 μm), curved and becomes chainform by aging.

(2) Gram's staining: negative or indefinite

(3) spore forming ability: yes

(5) motility: yes

(5) shape and characteristic of colonies: circular, corrugated orslightly convex, having smooth surface and transparent.

(6) growing temperature: grows at 70° C., but does not grow at 80° C.

(7) catalase: negative

(8) oxidase: negative

(9) O/F test: negative

(10) biochemical test:

Decomposes glucose, fructose, sorbose, D-arabinose, L-arabinose, ribose,D-xylitol, L- □xylitol, D-turanose, L-turanose, D-lyxose, D-tagatose,5-ketogluconic acid.

Does not decompose glycelol, erythritol, adonitol, β-methyl-D-xylose,galactose, mannose, rhamnose, dulcitol, α-methyl-D-mannose,α-methyl-D-glucose, N-acetyl-glucosamine, amidagline, arbutin,aesuculin, salicin, cellobiose, maltose, milk sugar, melibiose, canesugar, trehalose, inulin, melezitose, raffinose, glycogen, xylitol,gentiobiose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-ketogluconicacid.

Enzyme activity

β-galactositase negative arginine dihydrolase negative lyginedecarboxylase negative urease negative tryptophan deaminase negativegelatinase positive Others use of citric acid no production of H2S noproduction of indole negative production of acetoin positive reductionof nitrate positive anaerobic growth slightly observed growth at pH 7 nogrowth at pH 5.1 yes growth at 30° C. no

From above mentioned taxonomic features, the taxonomic positioning ofthis bacteria is referred in Bergey's Manual of Systematic Bacteriology,vol 2 p1104-1139, author: S. H. Sneath, editor: P H. Snerth et al.(publisher Williams & Willkins).

This bacteria is a spore-forming rod-shaped bacterium. AlthoughGram-negative nature of the bacterium is distinct from Gram-positivenature of known species of the genus Bacillus, it is recognized that itis a strain of the genus Bacillus because it grows aerobically.

Among known species of the genus Bacillus, B. acidocaldarius, B.lichenformis, or B. coagulans are known to be thermophilic andacidophilic. However, this strain should not be B. lichenformis and B.coagulance because B. lichenformis and B. coagulance arecatalase-positive and can grow at 40° C. but not at 65° C. Also, it isdifferent from the standard species of B. acidocaldarius because itproduces acetoin. Therefore, it is not possible to confirm that whetherit is a modified species of B. acidocaldarius or it belongs to adifferent species; the species of this bacteria can not be specified.

Example 2

5 ml of medium (pH 4.8) containing 1.5% of glucose, 1.5% of gelatin and0.01 % of yeast extract is poured into 5mi test tube and sterilized for20 minutes at 120° C. NTAP-1 (the shortened name of Bacillus genusNTAP-1 to discriminate the microorganism of this invention) isinoculated on said medium and cultivated with shaking for 4 days. Theculture medium is centrifuged for 20 minutes at 8,000 r.p.m., and theactivity of the thermostable collagen-decomposing enzyme in thesupernatant is measured. Namely, 0.4 ml of enzyme liquid is mixed to 0.1ml of 1M sodium acetate buffer (pH 4.5) and the mixture is pre-incubatedfor 5 minutes at 60° C. Then 3mg of Azocoll (azo dye-linked collagenpowder: product of Sigma Co., Ltd.) is suspended, and enzyme reaction iscarried out at 60° C. with stirring for 1 hour. After the reaction, thereaction mixture is chilled on ice, and insoluble Azocoll was separatedby centrifugation.

During the enzyme reaction, Azocoll is decomposed by the enzyme and thesupernatant turns red. By measuring the absorbance at 518 nm of thesupernatant, the activity of the thermostable collagen-decomposingenzyme is estimated. The amount of enzyme which makes the absorbance at518 nm increases 0.001 by 1 minute under said condition is defined as 1unit (U). The concentration of enzyme activity of the obtainedsupernatant liquid of the cultivated liquid is 3.1 U/ml.

Example 3

6 litter of same medium to Example 2 is poured into a jar fermentor of10-liter vessel. After sterilized for 30 minutes at 120° C., 200 ml ofthe NTAP-1 culture is inoculated on said medium. The cultivation iscarried out at 60° C. with 6 liters/min aeration for 4 days. Thethermostable collagen-decomposing activity of the culture supernatant ismeasured. The activity of enzyme of the supernatant liquid is 5.0 U/ml.

The resultant supernatant was used as the starting material, thepurification and concentration of thermostable collagen-decomposingenzyme was carried out according to the following process.

Ammonium sulfate is added to the supernatant liquid and the precipitateformed by ammonium sulfate 40% saturation was collected and dissolved in585 ml of 0.01M acetate buffer (pH 5.0). Phenyl-Sepharose (product ofAmeshamPharmacia Biotech.) was added to the solution, stirred and mixedfor 1 hour, then the mixture was filtrated and the resin is separated.The enzyme activity absorbed to the resin was eluted by washing theresin with 1:1 mixture of 0.01M acetic acid buffer (pH 5.0) and ethyleneglycol, and the active fraction was then dialyzed against 0.01Mphosphate buffer (pH 7.0).

Then, the solution was passed through DEAE-Sephadex (AmeshamPharmaciaBiotech) column which is previously equilibrated with 0.01M phosphatebuffer (pH 7.0). The linear gradient (0-1M) of sodium chloride was usedto elute the enzyme activity from the column. The eluate was fractionedinto about 90 fractions, and the activity of enzyme of each fractionsare measured according to the method described in Example 1.

Then, ammonium sulfate is added to the active fractions (233 ml) to 20%saturation. The enzyme activity is absorbed to a column by pass theenzyme solution through the phenyl-Sepharose (AmeshamPharmacia Biotech)column which is previously equilibrated with 0.01M acetic acid buffer(pH 5.0).

Then, the enzyme activity adsorbed to the column was eluted by washingthe column with a linear concentration slope (0-50%) of ethylene glycolin the equilibration buffer. The eluted solution is divided into 90fractions approximately, and the enzyme activity of each fraction ismeasured according to the measuring method described in Example 1. Fromfractions of eluted solution, active fractions (total 48 ml) arecollected.

This solution is dialyzed against 0.01M phosphate buffer (pH 7.0), thenapplied to a column of MONO-Q (AmeshamPharmacia Biotech) which ispreviously made equilibrated with the same buffer. The activity waseluted by washing the column with a linear gradient (0-1M) of sodiumchloride.

Active fractions (10.6 mi) were collected and concentrated to 0.5 mlusing Centricon (centrifuge concentrator: Amicon Co., Ltd.: saidconcentrator has an ultrafiltration membrane made of cellulosederivative at the bottom of the container. When mixed solution composedof enzyme and protein is poured into the container and centrifuged by6000 r.p.m., protein contained in the solution is remained on themembrane, while water or low molecular weight ion passes through thefilm and recovered as a filtrated liquid.), and divided by a gelfiltrating chromatography method, and 1.2 ml of fraction having higheractivity is collected. The activity yield of the dissolved fraction fromthe cultivated liquid is 1.4%, and the concentration of the activatedenzyme is 416 U/ml.

The enzyme solution thus obtained was analyzed by SDS-polyacrylamide gelelectrophoresis according to Laemmli procedure (Laemmli, U.K., Nature,1970, 227, 680-685), and molecular weight of the thermostablecollagen-decomposing enzyme is estimated to be approximately 46,000.

Example 4 Experiment to Investigate the Substrate Specificity of theThermostable Collagen-decomposing Enzyme

After 5 mg of Azocoll is suspended in 0.4 ml of 0.01M sodium acetatebuffer (pH 4.5), 0.1 ml of enzyme solution, whose enzyme concentrationis adjusted properly by dilution, is added and allowed to react for 1hour at 60° C. with constant shaking. After the reaction, the reactionmixture was chilled on ice for 1 hour and then centrifuged. Theabsorbance at 518 nm of the supernatant is measured. The reactionmixture prepared by same process except using water instead of enzymesolution was used as a blank solution. The absorbance at 518 nm of thereaction mixture whose added Azocoll was perfectly solubilized ismeasured. The same experiments are carried out on various enzymeconcentrations, and the amount of enzyme which gives approximately 50%degradation (to solubilize approximately 2.5 mg Azacoll) under thesecondition is determined.

By the same process as mentioned above, 5 mg of collagen, gelatin,casein or cow serum albumin (all are the products Nacalai Tesque, Co.)are respectively suspended (or dissolved) in 0.4 ml of 0.01 M sodiumacetate buffering solution (pH 4.5), then 0.1 ml of enzyme solution ofpreviously decided concentration is added and reacted for 1 hour at 60°C. with constant shaking. After the reaction, the reaction mixture waskept at 4° C. for 20 minutes and centrifuged. in cases which use caseinor bovine serum albumin, 0.5 ml of 50% trichloroacetic acid is added tothe reaction mixture and was kept at 4° C. for 20 minutes thencentrifuged. The adsorption at 280 nm of the supernatant was measured.From the adsorption value at 280 nm when each proteins are perfectlysolubilized, the decomposing ratio of each proteins are measured. Whenthe decomposing rate of Azocoll is regarded as 100%, the relativesolubilizing rate of each proteins are listed in Table 1.

TABLE 1 Relative solubilizing rate of each proteins, when decomposingrate of Azocoll is regarded as 100% collagen +++ gelatin +++ casein ±albumin ± keratin − remarks +++: has relative activity greater than 80%to Azocoll ±: has relative activity lower than 20% to Azocoll −: notreacted

The obtained results are shown in FIG. 1. This enzyme is stable up to60° C. In the meanwhile, the stability of enzymes when enzymes aretreated by various pH are investigated. The buffer to be used inspecific pH range are listed below.

pH 2.5 to 3.5: 1M glycine-HCl buffer

pH 3.5 to 5.5: 1M sodium acetate buffer

pH 6.0 to 8.0: 1M sodium phosphate buffer

pH 8.0 to 9.0: 1M glycine-NaOH buffer

pH 9.0 to 10.0: 1M sodium phosphate buffer

After 0.0025 ml of these buffers and 0.0025 ml of 1% aqueous solution ofTween 80 are added to 0.02 ml of enzyme liquid, placed at thetemperature of 60° C. for 1 hour. Then, 0.05 ml of 1M acetic acid buffer(pH 4.0) is added to this treated enzyme liquid and the activity ismeasured. The obtained result indicates that this enzyme is stable atthe pH range from 3 to 6 (FIG. 2).

Example 7 Experiment to Investigate the of the Optimum ReactionTemperature and Optimum Reaction pH of Thermostable Collagen-decomposingEnzyme

The activity of the thermostable collagen-decomposing enzyme of thisinvention was measured at 30, 40, 50, 60, 70 and 80° C. The methoddescribed in Example 1 was used except changing the reactiontemperature. The results showed that this enzyme exhibited the highestactivity at 60° C. (refer to FIG. 3).

Secondly, the activity of this enzyme is measured at various (from pH2.5 to 7.2). The method for activity measurement was based on the methoddescribed in Example 1 except changing buffer component to be used inthe reaction system as follows: 0.01M glycine-HCl buffer (pH 2.5 to7.2), 0.01M sodium acetate buffer (pH 4 to 6) or 0.01M potassiumphosphate buffer (pH 6 to 8). The results showed that this enzyme showedthe highest activity at pH 3. 7 to 3.9 (refer to FIG. 4).

POTENTIALS FOR THE INDUSTRIAL USE

Obviously from the above mentioned Examples, by the present invention,it becomes possible to prepare effectively a thermostablecollagen-decomposing enzyme which is excellent at the optimumtemperature, optimum pH and collagen substrate specificity by the use ofabove mentioned novel microorganism. Therefore, the present inventionmake it possible to utilize the materials which are not utilized inlivestock industries, and largely contribute to the production ofcollagen peptides that has potential applications in medical,pharmaceutical, and food industries.

What is claimed is:
 1. A substantially purified or isolated thermostablecollagen-decomposing enzyme obtained from bacillus genus bacteria strainNTAP-1, having a molecular weight of approximately 46,000 Da, an optimumreaction pH between pH 3.5 and 4.5, an optimum reaction temperaturebetween 65° C. and 70° C., wherein the enzyme retains more than 60% ofits original activity after heat treatment at 60° C. and pH 6.0 for 4hours and wherein the enzyme is stable between pH 3 to
 6. 2. A methodfor producing the substantially purified or isolated thermostablecollagen-decomposing enzyme described in claim 1 comprising the steps ofculturing bacillus genus bacteria strain NTAP-1, deposited underaccession number FERM BP-6926, and collecting the resulting thermostablecollagen-decomposing enzyme.
 3. A substantially purified or isolatedmicroorganism NTAP-1, deposited under accession number FERM BP-6926,which produces the thermostable collagen-decomposing enzyme of claim 1.4. The substantially purified or isolated thermostablecollagen-decomposing enzyme of claim 1, wherein said enzyme decomposescollagen and gelatin at a first decomposing rate greater than 80%relative to that of Azocall, and decomposes casein and albumin at asecond decomposing rate lower than 20% relative to that of Azocall.